Plasmid pBUL1 derived from a lactobacillus and derivatives thereof

ABSTRACT

Disclosed is the plasmid pBUL1 having a restriction endonuclease cleavage map as shown in FIG. 1 and having a length of about 7.9 kbp and its derivatives. The plasmid was isolated from Lactobacillus delbrueckii subsp. bulgaricus M-878. The plasmid is useful as a vector for breeding various microorganisms such as lactic acid bacteria, and the derivatives thereof are useful also as a shuttle vector.

This is a continuation of parent application Ser. No. 07/940,852 filedOct. 22, 1992 filed as PCT/JP92/00193 Feb. 24, 1992 now U.S. Pat. No.5,426,049.

FIELD OF THE INVENTION

The present invention relates to a novel circular double-stranded DNAplasmid pBUL1 derived from Lactobacillus delbrueckii subsp. bulgaricus,the utility and safety of which as a yogurt-producing bacterium are wellknown, and to derivatives thereof, as well as to microorganismstransformed with the plasmids.

PRIOR ART AND PROBLEMS TO BE SOLVED BY THE INVENTION

Lactic acid bacteria are very useful microorganisms which have long beenused in production of various fermented foods. If a recombinant DNAtechnology which has rapidly been developed in recent years could beapplied to lactic acid bacteria, greater enhancement of the utility ofthe bacteria would be expected. In fact, host-vector systems with afairly high efficiency have already been reported for lactic acidbacteria of some species of, for example, Lactococcus lactis (reference1), Streptococcus salivarius subsp. thermophilus (reference 2),Lactobacillus plantatum (reference 3) and Lactobacillus casei (reference4), and the current stage of the technical improvement is use thehost-vector systems on an industrial scale.

However, despite great efforts by many researchers, there has hithertobeen no report of transformation of Lactobacillus delbrueckii subsp.bulgaricus and Lactobacillus delbrueckii subsp. lactis (hereinafterreferred to as Lb. bulgaricus, and Lb. lactis, respectively), which areused extensively as bacteria for producing milk products such as yogurt.Application of broad-host-range plasmids, such as pNZ12 (reference 5),pGK12 (reference 6) and pIL253 (reference 7) with which transformants oflactic acid bacteria of some species have been obtained, to theabove-mentioned two subspecies has been attempted, but transformationwas not successful. However, establishment of host-vector systems of theabove-mentioned two subspecies has been earnestly desired;

If a recombinant DNA technology is applied to microorganisms to be usedin production of foods, the safety of vectors to be used fortransformation of them must be established. As vectors, for this purposethose vectors are desirable which exist naturally in microorganisms thathave been used to produce foods which have traditionally been eaten andthe safety of which has been historically confirmed. On the other hand,fermented milk products such as yogurt are foods which have been eatenfor a long period of time and their safety has been well confirmed.Therefore, the plasmids derived from microorganisms from fermented milkproducts, for example, those of the above-mentioned two subspecies, areuseful vectors in order to construct transformants of microorganims forfood production. In addition they are also available vectors fortransformation for the purpose of producing various physiologicallyactive substances.

Means for Solving the Problems

For the purpose of developing host-vector systems for Lb. bulgaricus andLb. lactis, the present inventors variously investigated and studiedplasmids of these subspecies. As a result, they are the first who havesucceeded in isolating a plasmid from Lb. bulgaricus M-878 strain (FERNBP-3758) possessed by Meiji Institute of Health Science of Meiji MilkProducts Coo, Ltd., which has a length of about 7.9 kbp, which has arestriction endonuclease map as shown in FIG. 1, and which does not haverecognition sites for BamHI, EcoRI, KpnI, PstI and SalI, the basesequence of the SmaI fragment with 1344 bp being represented as theSequence Number 1 of the Sequence Table in Table 1 below. They named theplasmid pBUL1.

                                      TABLE 1                                     __________________________________________________________________________    (SEQ ID NO: 1)                                                                Sequence Table:                                                               Sequence Number: 1                                                            Length of Sequence: 1344                                                      Type of Sequence: Nucleic Acid                                                Number of Strand: Double-stranded                                             Topology: Linear                                                              Kind of Sequence: Other Nucleic Acid                                          Origin:                                                                       Name of Microorganism: Lactobacillus delbrueckii                              subsp. bulgaricus                                                             Name of Strain: M-878                                                         Sequence:                                                                     __________________________________________________________________________    CCCGGGGCGA                                                                            AACGACATGG                                                                            GGCGCTCAAA                                                                            CCATTGCTGA                                                                            GGCGATCAAT                                                                            TACGTGCAAG                                                                            80                            CCCAGCATCC                                                                            CGATCACGGC                                                                            TATTTCCCAG                                                                            CTCGCCAAAA                                                                            TTCCGGCATG                                                                            AGGGTTGTTG                                                                            120                           AACCGGGTGA                                                                            AAGAGCCACA                                                                            GGCGAAACGC                                                                            TTAGAATTAC                                                                            GATTGACGGA                                                                            CAGGAAAGAG                                                                            180                           AATTTCCGTT                                                                            CAACGGCTTT                                                                            TTCTATAACC                                                                            GGGATTATGA                                                                            AATGACCGAG                                                                            GTTGGGTTTG                                                                            240                           CTAACAGGTT                                                                            TGCCGATTGG                                                                            TACGCCAAAG                                                                            GGAAACTTGT                                                                            TTATCACCCC                                                                            GGCTTAAAGG                                                                            300                           CGTGGCTTAT                                                                            GTACAACCCA                                                                            GAAACGGGGT                                                                            CATGGATGCC                                                                            GAATGAAGAC                                                                            GACAGACTGG                                                                            350                           GCAAGGATTT                                                                            TAACCAGACC                                                                            CCGGAAAAAC                                                                            TCATCGATAA                                                                            CTTGCGGATA                                                                            AACCTCAAAT                                                                            420                           TTGAACAACC                                                                            GCTATGGAAA                                                                            AAAGTTGGGT                                                                            TTAATCCCCA                                                                            AAAGCCTAAT                                                                            AATCAAACAT                                                                            480                           TCGGGGAAAA                                                                            GGCTTATGCT                                                                            AGTGGCTATA                                                                            GCCGGATCAG                                                                            CACGGCCGCT                                                                            GGGCAAAAGG                                                                            540                           CGACCCTTGA                                                                            ATTAGCTCAG                                                                            AGCCGTTTAA                                                                            CCGTGCGTGC                                                                            ATTTAACGAC                                                                            TGTAAGACCG                                                                            600                           CGGCGAAACT                                                                            TTTTGACAAG                                                                            GTCACGGATG                                                                            CTGGTTTGCC                                                                            TAATAAAGCC                                                                            ACAGAGGGTG                                                                            720                           ATGGCGGCAA                                                                            GCTCTGGGAT                                                                            CGTTTCCTGA                                                                            AAGAGACCTT                                                                            TTGCGGCGAT                                                                            CTTGATTTGA                                                                            780                           TCGAGTTCGT                                                                            ACAGGCCTGC                                                                            ATAGGCTACA                                                                            GCATTACGGG                                                                            CAAAATCAAT                                                                            GAACAGGTCA                                                                            840                           TGTTTATCTG                                                                            CAAAGGCAGT                                                                            GGGGGCAACG                                                                            GGAAGAGCAT                                                                            TTTTCTTGAA                                                                            TGCTTAAACG                                                                            900                           AGGTGCTGGG                                                                            CGATTATAGC                                                                            TCTGTTATCC                                                                            CAATAGAAAC                                                                            GCTAACAGAC                                                                            AACGGCAAGG                                                                            960                           CTCAGCGTGA                                                                            CGGATCAGCA                                                                            CCAAGTCCAG                                                                            ACCTTGCAAG                                                                            CCTTGAGGGC                                                                            AAGCGGTTCG                                                                            1020                          TTATTACGAG                                                                            CGAACCGAAA                                                                            GAGCAGGTTA                                                                            CAATCGATGC                                                                            TGGGACGGTC                                                                            AAAACGGTGA                                                                            1080                          CGGGTGGCAC                                                                            TAAGTTAAAA                                                                            GTTAGAATGC                                                                            TACACCAAAA                                                                            CCCGATTGAG                                                                            TTCCTGCCAC                                                                            1140                          AGTTTAAAAT                                                                            TTGGTGGCAA                                                                            TCTAACGGCT                                                                            TGCCAAACGT                                                                            CAACTTTAAC                                                                            GATTATGCTA                                                                            1200                          TTTTACGGCG                                                                            CTTGATCGTC                                                                            ATCCCGTTTA                                                                            AAAATGAGGT                                                                            GCGAGAGGAT                                                                            GCGGTAGATA                                                                            1250                          TCAACCTCAA                                                                            AAGCAAGCTA                                                                            ATGAAAGAGA                                                                            AAGAGTTTAT                                                                            TTTAAAGTGG                                                                            TGTGTTGAGG                                                                            1320                          GCGTGGCTAA                                                                            ATGGCAAGCC                                                                            CGGG                            1344                          __________________________________________________________________________

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1:

This is a restriction endonuclease map of pBUL1. The recognition site ofeach restriction enzyme is expressed by kbp unit, on the basis of BglII.pBUL1 does not have recognition sites for BamHI, EcoRI, KpnI, PstI andSalI. In the structure of pBUL1, the region necessary for replication ofpBUL1 is shown by the thick line; and the region corresponding to thebase sequence of the Sequence Number 1 (the third largest fragment ofall SmaI fragments) is designated as "1344 bp SmaI fragment" in theouter periphery of the map.

FIG. 2:

This shows a scheme of construction of the erythromycin-resistancecassette plasmid p8Em1, in which Δ indicates the pUC118-derived multiplecloning site.

FIG. 3A-3D:

This shows restriction endonuclease maps of the derivatives of pBUL1,i.e., pX3, pX4, pS3 and pS4 (Although all of these plasmids arecircular, they are represented as linear profiles on the basis ofBglII.). In the drawing, the fine lines each indicate the sequence ofpBUL1, and the thick lines each indicate the pAMβ1-derivederythromycin-resistance gene.

FIG. 4A-4B:

This shows restriction endonuclease maps of plasmids pU8ST8 and pU8ST9.In the drawing, the striped arrows each indicate the L-lactatedehydrogenase gene of Streptococcus salivarius subsp. thermophilus M-192strain (ST-LDH gene); and the fine lines each indicate the sequence ofplasmid pUC118. bla indicates the ampicillin-resistance gene.

FIG. 5:

This shows restriction endonuclease maps of plasmids pXL38, pXL39, pXL48and pXL49 (Although all of these plasmids are circular, they arerepresented as linear profiles on the basis of BglII.). In the drawing,the fine lines each indicate the sequence of pBUL1, the thick lines eachindicate the pAMβ1-derived erythromycin-resistance gene, and the stripedarrows each indicate ST-LDH gene.

FIG. 6:

This shows a scheme to locate the region required for replication ofpBUL1 by the deletion method.

FIG. 7:

This shows a restriction endonuclease map of pBR3Δ18E1 plasmid byligation of pX3Δ18E plasmid with the E. coli plasmid (pBR322). In thedrawing, the fine line indicates the sequence derived from plasmidpBR322, the thick line indicates the sequence derived from pX3Δ18E, thearrow (Em^(r)) indicates the erythromycin-resistance gene, and the arrow(Ap^(r)) indicates the ampicillin-resistance gene.

FIG. 8:

This shows a restriction endonuclease map of pBR3Δ18E2 plasmid. Thesymbols are the same as those, shown in FIG. 7.

FIG. 9:

This shows a restriction endonuclease map of p8X3Δ18E1 plasmid byligation of pX3Δ18E plasmid with the E. coli plasmid (pUC118). in thedrawing, the fine line indicates the sequence derived from plasmidpUC118, the thick line indicates the sequence derived from pX3Δ18E, thearrow (Em^(r)) indicates the erythromycin-resistance gene, and the arrow(Ap^(r)) indicates the ampicillin-resistance gene.

FIG. 10:

This shows a restriction endonuclease map of p8X3Δ18E2 plasmid. Thesymbols are the same as those shown in FIG. 9.

Since the phenotype encoded by the pBUL1 was cryptic, anerythromycin-resistance gene was added to the plasmid as a selectivemarker in transformation experiments (Erythromycin may be referred tosimply as "Em" hereunder.). As a result, the present inventors havesucceeded in obtaining transformants expressing the above-mentionedselective marker in microorganisms of three genera, namely Bacillussubtilis, Lactococcus lactis subsp. lactis and Lactobacillus delbrueckiisubsp. lactis. The fact indicates that the plasmid pBUL1 (hereinafteroften referred to as "the plasmid of the invention") has a broad hostrange in Gram-positive bacteria. The present invention has beencompleted on the basis of these findings.

Since the gene which participates in self-replicatability of pBUL1 isconsidered to be encoded in a part of the plasmid DNA of the presentinvention as reported in other plasmids, any other plasmid derivativesderived either by deletion of unnecessary regions for replication fromthe plasmid of the invention, or by insertion or addition of any otherDNA to pBUL1 are also considered to have the same function as theplasmid of the invention. For instance, as described in Example 6followed hereinafter, any plasmid causes no hindrance in replication, ifit contains, as a region necessary for replication, a region necessaryfor replication in about 4 kbp DNA fragment indicated by the thick linein FIG. 1 between the recognition site for Eco47III and the positionabout 1.1 kbp apart clockwise from the ScsI site, namely about 0.45 kbpapart counterclockwise from the NdeI site. Therefore, the presentinvention is not restricted to only the plasmid pBUL1 itself butincludes other derivative plasmids as obtained by modifying pBUL1 aswell as other recombinant plasmids as obtained by inserting othergene(s), for example, marker(s) such as Em-resistance gene or exogeneousgene(s) such as L-lactate dehydrogenase gene, or other promoter(s) oroperator(s) into it.

As derivative plasmids of pBUL1 of the present invention, for example,there are mentioned plasmids containing necessary regions forreplication in the region of about 4 kbp shown as the thick line in FIG.1 as mentioned above. As one example of them, there is the mentionedplasmid pX3Δ18E (FIG. 6, the bottom). This plasmid can be replicated notonly in Gram-positive bacteria such as lactic acid bacteria but also inE. coli, as is obvious from Example 8. This plasmid itself is nothingbut a plasmid with a broad-host-range, which has been desired in thistechnical field. Since this plasmid is one which can be replicated notonly in Gram-positive bacteria (e.g., lactic acid bacteria, Bacillussubtilis) but also in Gram-negative bacteria (e.g., E. coli), it iseffective as a novel versatile shuttle vector which has extremely highapplicability and can be used in both Gram-positive bacteria andGram-negative bacteria.

Another example of the pBUL1-derived plasmids can be obtained byligation of other plasmids (fragments) with the region of pBUL1necessary for replication, involved in the region of about 4 kbp shownas the thick line area in FIG. 1, or a DNA fragment of pBUL1 whichcontains this region. As the other plasmids to be ligated, variousplasmids may be used widely. As some examples of them, there areexemplified E. coli-derived plasmids such as pBR series plasmids and pUCseries plasmids.

Also the thus ligated plasmids have been identified to be replicable inboth lactic acid bacteria and E. coli, like the above-mentioned plasmidpX3Δ18E, and they may also be used as plasmids with a broad-host-rangewhich have heretofore been strongly desired in this technical field oras shuttle vectors between Gram-positive bacteria and Gram-negativebacteria. As examples of such ligated plasmids, there are exemplifiedpBR3Δ18E1, pBR3Δ18E2, p8X3Δ18E1, p8X3Δ18E2 (FIGS. 7 to 10), etc.

As mentioned above, plasmid pBUL1 and its derivative plasmids show abroad-host-range and are useful. Using them as a vector, expression ofheterogeneous genes in lactic acid bacteria is possible.

For instance, where a recombinant plasmid was constructed by ligation ofpBUL1 plasmid with an erythromycin-resistance gene as a selective markergene and an L-lactate dehydrogenase gene derived from a yogurtlactococcus (Streptococcus salivarius subsp. thermophilus); (e.g., pXL38in FIG. 5) as an exogenous gene, and the recombinant plasmid wasintroduced into Lb. lactis which does not naturally have L-lactic acidproducibility, the resulting erythromycin-resistance transformantproduced not only D-lactic acid but also almost the same amount ofL-lactic acid as metabolic end-products.

Thus, plasmid pBUL1 and its derivative plasmids are useful also as avector for expression of heterogeneous genes in lactic acid bacteria.

For preparing the plasmid pBUL1 of the present invention, Lb. bulgaricusM-878 strain is first cultivated in a liquid medium which is used forcultivation of lactic acid bacteria, for example, LCM medium (reference8), according to ordinary methods of cultivating lactic acid bacteriaunder ordinary incubation conditions. Next, the incubated cells arecollected and are then subjected to lysis by known methods for lacticacid bacteria, for example, by using an enzyme such as lysozyme ormutanolysin, etc. From the resulting cell lysate, the intended plasmidcan be isolated and purified by a conventional emplyed method, such asphenol extraction and cesium chloride density gradient centrifugation inthe presence of ethidium bromide. For construction of the derivativeplasmids of pBUL1, which are other plasmids of the present invention,the plasmid pBUL1 may be treated for digestion, ligation and others byknown methods (reference 10).

In order to transform microorganisms by introducing thereinto arecombinant plasmid as obtained by inserting a selective marker gene tothe plasmid pBUL1, a known method which is considered to be the best forthe host may be selected from conventional methods, such as calciumchloride method, protoplast-polyethylene glycol method, electropotationmethod, etc., with no particular limitation, according to thecharacteristics of microorganisms to be transformed. Where lactic acidbacteria are used as a host, the electropotation method is preferred. Amarker for selecting the transformants may be selected from variousantibiotic-resistance genes known in this technical field. Where thetransformed microorganisms are intended to be used in producing foods ormedicines, use of markers which have been confirmed to be safe isdesirable. If desired, other base sequences participating in control ofexpression, such as promoters, may also be inserted into the plasmids ofthe invention.

Recombinant plasmids are considered to be safe which are constructedfrom pBUL1 plasmid or its derivative, an enzyme gene derived from amicroorganism used in food production and a safe selective marker gene.Therefore, the transformants of food-producing bacteria such as lacticacid bacteria with such a safe plasmid are also considered to be safe.Such safe transformants may be incubated by ordinary methods to producea large amount of enzymes and physiologically active substances, andthey may be used in various food productions in ordinary ways to attainthe intended objects. In addition, since the safety of the transformantsis highly assured in any case, they do not cause by-production of anybiohazards or harmful substances. Accordingly, it is expected that theycould advantageously be utilized in industrial production of medicinesand foods which especially need safe methods of production.

Next the present invention will be explained hereunder by way of thefollowing examples.

EXAMPLE 1

(Preparation of Plasmid pBUL1)

Lactobacillus delbrueckii subsp. bulgaricus M-878 strain (FERM BP-3758,as possessed by Meiji Institute of Health Science; herein often referredto simply as M-878 strain) was subcultivated with a skim milk medium(liquid medium as prepared by dissolving 10% skim milk powder and 0.1%yeast extract in distilled water and sterilized at 121° C. for 7minutes) and inoculated (0.5%) in 6 liters of LCMG medium (as preparedby adding 1% (w/v) glucose to LCM medium) and incubated at 37° C. for 15hours.

After the incubation, the cells were Collected by centrifugation andwashed twice with 20 mM Tris-HCl buffer (pH 7.0). The washed cells weresuspended in 480 ml of a hypertonic buffer (20 mM Tris buffer containing0.3M raffinose, 5 mM magnesium chloride and 5 mM calcium chloride; pH7.0). To this were added mutanolysin and lysozyme in an amount of 5μg/ml and 500 μg/ml, respectively, as the final concentrations, and wereincubated at 37° C. for 10 minutes. Then, 54 ml of 250 mM EDTA (pH 8.0)was added to the resulting solution, which was then subjected tocentrifugation to collect the precipitates.

The precipitates were then suspended in 240 ml of 50 mM Tris buffer (pH8.0) containing 6.7% (w/v) sucrose and 25 mM EDTA. The resultingsuspension was then processed according to Anderson & Mckay method(reference 9), from lysis with SDS to rough purification of plasmid DNA.

The crude plasmid DNA preparation thus obtained was subjected to RNasetreatment by an ordinary method (reference 10) and then to cesiumchloride density gradient centrifugation in the presence of ethidiumbromide to obtain about 1 μg of purified pBUL1 plasmid DNA.

The pBUL1 plasmid was cut with various commercially availablerestriction enzymes, and the length of each fragment obtained wascalculated after agarose gel electrophoresis. As a result, pBUL1 wasidentified to be a circular double-stranded DNA plasmid having therestriction endonuclease map in FIG. 1 with a total length of about 7.9kbp. pBUL1 did not have recognition sites for BamHI, EcoRI, KpnI, PstIand SalI. Of five fragments obtained by digestion of pBUL1 with SmaI,the third largest fragment (1344 bp; the position of which has beendesignated in the outer periphery of the restriction endonuclease map inFIG. 1 as "1344 bp SmaI fragment") was analyzed with respect to the basesequence thereof, which is shown as Sequence Number 1 in Table 1.

EXAMPLE 2

(Addition of Selective Marker Em-resistance Gene to pBDL1)

First, conjugatively transmissible plasmid pAM β1 (reference 11) derivedfrom Enterococcus faecalis was cut with HhaI and subjected to agarosegel electrophoresis to thereby cut out a gel fraction containing DNAfragments ranging about 1.1 kbp having the Era-resistance gene. DNAswere isolated from the thus cut-out gel using a GENECLEAN DNA purifyingkit (product by BI0101 Co.). The DNA fragment was ligated to E.coli-derived pUC118 plasmid (product by Takara Shuzo Co.) according tothe process shown in FIG. 2 to prepare "cassette plasmid" p8Em1 in orderto excise the Em-resistance gene successfully with various restrictionenzymes.

Next, about 0.25 μg of p8Eml DNA was cut with XbaI and was ligated witha fragment as obtained by cutting about 0.025 μg of pBUL1 DNA with XbaI.A half of the reaction mixture after the ligation was used intransformation of Bacillus subtills 207-25 strain (reference 13) by themethod of Chang et al (reference 12). One third of the transformed cellswere spread on a plate of DM3 medium containing 25 μg/ml of erythromycinand incubated for 2 days at 37° C. to obtain Em-resistant transformants.

Plasmid DNAs were prepared from the transformants obtained, and therestriction endonuclease cleavage pattern of the plasmids was analyzed.When pBUL1 and the Em-resistance gene were ligated at XbaI site, fivetransformants of all the nine analyzed contained a plasmid having therestriction endonuclease map of A in FIG. 3; and two of them contained aplasmid having the restriction endonuclease map of B in FIG. 3. Theplasmid with the map A is FIG. 3 was named pX3; and that with the map Bin the same was named pX4.

In the same manner as above, except that p8Eml was cut with SmaI andpBUL1 was cut with ScaI, and the resulting fragments were subjected toblunt end ligation followed by transformation of Bacillus subtilis207-25 strain with the ligated products, Em-resistant transformants werealso obtained. Five of the six transformants, analyzed with respect toplasmids therein, contained a plasmid having the restrictionendonuclease map of C in FIG. 3; and one of them contained a plasmidhaving the restriction endonuclease map of D in FIG. 3. These plasmidswere named pS3 and pS4, respectively.

As mentioned above, recombinant plasmids pX3, pX4, pS3 and pS4 (allhaving a length of about 9.0 kbp) were constructed by introducing thepAMβ1-derived Em-resistance gene (having a length of about 1.1 kb) topBUL1. In addition, it was shown that pBUL1 could function as a plasmidreplicon in Bacillus subtilis.

EXAMPLE 3

(Transformation of Lactococcus lactis subsp. lactis):

Lactococcus lactis subsp. lactis (hereinafter often referred to simplyas "Lc. lactis") IL1403 strain (as obtained from Dr. Alain Chopin, INRA,France) was used. To this was introduced pBUL1 having the Em-resistancegene, whereby a transformant of Lc. lactis showing Em-resistance wassuccessfully obtained. The details are mentioned below.

From the transformants of Bacillus subtills obtained in Example 2, therecombinant plasmids, PX3 and pX4, constructed by inserting theEm-resistance gene into pBUL1 were prepared by the method of reference14.

Next, pX3 and pX4 each were introduced into Lc. lactis IL1403 strainaccording to the method of reference 1. The transformants were obtainedby selecting on agar plates of BL medium (product by Eiken Chemical Co.)containing 25 μg/ml of Em.

The transformants were incubated in LCMG medium, and plasmids wereobtained from the cells after Anderson et al. (reference 9). Theplasmids thus obtained were shown to have the same restriction enzymerecognizing sites as those of the plasmids used for the transformation.From the result, it was concluded that pBUL1 also functioned as aplasmid replicon in not only Bacillus subtills but also Lc. lactis.

EXAMPLE 4

(Transformation of Lactobacillus delbrueckii subsp. lactis):

From the transformants of Lactococcus lactis subsp. lactis IL1403obtained in Example 3, plasmids pX3 and pX4 were prepared. Using them,Lb. lactis ATCC 12315 strain and M-908 strain, possessed by MeijiInstitute of Health Science, were transformed by electroporation. Therewas no report of success in transformation of Lactobacillus delbrueckiispecies being commercially useful, despite studies by many researchers.Using the plasmid the invention, transformation of Lb. lactis wasattained for the first time, as mentioned below.

Lb. lactis ATCC 12315 strain or N-908 strain as subcultivated in a skimmilk medium was inoculated in LCMG medium in a concentration of 2% andincubated for 2 hours at 42° C. The cells were collected and washed, andthen suspended in EP buffer (containing 0.4M sucrose, 1 ml magnesiumchloride and 7 ml potassium dihydrogenphosphate; pH 7.4) at aconcentration of OD₆₆₀ =4.0 and cooled on ice. 0.8 ml of the cellsuspension was put in an electroporation cuvette, about 0.1 to 2 μg ofpX3 or pX4 plasmid was added thereto, and an electric pulse of 25 μF at2.5 kV was discharged thereto with Gene Pulser (manufactured by Bio-RadCo.).

Immediately after the discharge, the cells were suspended in 4 ml of anexpression medium (LCMG medium to which 0.2M raffinose, 5 mM magnesiumchloride and 1% lactose had been added) and incubated statically for 2.5hours at 37° C. All the culture liquid thus incubated was poured intoseveral plates and 10 to 15 ml of BL agar medium (sterilized and kept at50° C.) containing 25 μg/ml of Em was added thereto and mixed. After theculture medium solidified, the plates were incubated anaerobically inGaspak system (manufactured by Beckton-Dickinson Co.) at 37° C. for 2 to4 days, and the transformants were selected. According to the method,about 10 to 100 transformants per μg of the plasmid DNA were obtained.

The Em-resistant clones thus obtained showed a strong Em-resistance (>1mg Em/ml) and had a plasmid having the same restriction endonuculeasemap as pX3 or pX4. From these results, they were confirmed to betransformants. Further, using the pX3 or pX4 plasmid DNA obtained fromthe transformants, the transformation frequency in Lb. lactis ATCC 12315strain increased by about 10 times.

No transformants of Lb. lactis have heretofore been obtained by usingother plasmids such as pGK12 or IL253 under the same conditions asabove. From the fact, it is obvious that the pBUL1 of the presentinvention is useful as a vector for Lb. lactis.

EXAMPLE 5

(Introduction and Expression in Lb. lactis of L-lactate DehydrogenaseGene inserted in pBUL1):

A restriction enzyme SspI fragment (about 1.2 kbp) containing a gene(Japanese Patent Application No. 2-45976) coding for L-lactatedehydrogenase (hereinafter referred to as "ST-LDH") derived from alactic acid bacterium Streptococcus salivarius subsp. thermophilus M-192strain (possessed by Meiji Institute of Health Science) was insertedinto the SmaI recognition site of pUC118, to construct the recombinantplasmids pU8ST8 and pU8ST9 as shown in FIG. 4-A and FIG. 4-B,respectively.

These recombinant plasmids were cut with BamHI and KpnI and subjected toagarose gel electrophoresis. About 0.02 μg of DNA fragments containingthe ST-LDH gene was cut out and isolated using a GENECLEAN DNApurification kit (manufactured by BI0101 Co.). These DNA fragments wereligated with about 0.3 μg of pX3 or pX4, obtained in Example 2, cut withBamHI and KpnI. Using this ligation mixture, Lc. lactis IL1403 strainwas transformed, whereby transformants having the plasmids as shown inFIG. 5 were obtained. These plasmids were named pXL38, pXL39, pXL48 andpXL49, respectively. These plasmids were prepared by the method ofreference 9 and were used for transformation of Lactobacillusdelbrueckii subsp. lactis ATCC 12315 strain by the same method as shownin Example 4. As a result, Em-resistant transformants were obtained.These carried plasmids each having the same restriction endonuclease mapas the introduced plasmids.

These transformants were incubated in a skim milk medium. The culturewas diluted with distilled water and subjected to centrifugation. Thelactic acid in the resulting supernatant was measured by the use of alactic acid measuring kit (F Kit L-lactic Acid; manufactured byBoehringer Mannheir). As a result, L-lactic acid, which is not naturallyproduced at all by the host, was detected in an amount almost equivalentto D-lactic acid produced.

The cells of one of the transformants were disrupted and the cellextract was prepared. As a result, L-lactate dehydrogenase activity,which is not detected in the host cells, was detected in the cellextract of the transformant. The L-lactate dehydrogenase from thetransformant was purified and the sequence of the 18 N-terminal aminoacids thereof was examined. As a result, it was identical to that ofST-LDH. From the above-mentioned results, it was shown that aheterogeneous gene expression was possible in Lb. lactis with pBUL1 as areplicon.

EXAMPLE 6

(Presumption of the Region in pBUL1 necessary for Replication by aDeletion Method)

pX3 was cut with BamHI and KpnI and subjected to deletion in thedirection as indicated in FIG. 6, by the use of a DNA deletion kit forkilosequencing (manufactured by Takara Shuzo Co.). Each reaction mixturewas applied to transform Lc. lactis IL1403 in the same method as inExample 3, and the transformants were selected in the presence of 25μg/ml of Em.

Plasmids harboured by the Em-resistant transformants were prepared andtheir restriction enzyme recognition sites were examined. As a result,the shortest deletant of pX6b 3 was obtained whose deletion proceeded tothe position in pX3 shown in FIG. 6, namely, the position about 1.1 kbpclockwise apart from ScaI site and about 0.45 kbp counterclockwise apartfrom NdeI site. The deleted plasmid was named pX3Δ18 and it was furthercut with both PstI and either of BglII, SphI and Eco47III and thensubjected to self-circularizing ligation. The reaction mixture was usedin transformation of Lc. lactis IL1403 strain and the selection of thetransformants was performed as in Example 3. As a result, transformantshaving plasmids of various sizes were obtained. The plasmid pX3Δ18E asshown in FIG. 6 is the shortest one, which was formed by cutting pX3Δ18with PstI and Eco47III followed by self-ligation. The region necessaryfor replication of pBUL1 was found to be contained in about 4 kbp DNAfragment of pX3Δ18E.

EXAMPLE 7

(Formation of Shuttle Vectors by Ligation of pX3Δ18E Plasmid with E.coli Plasmid)

pX3Δ18E plasmid obtained in Example 6 was prepared from Lc. lactisIL1403 strain by the method of reference 9. The thus obtained pX3Δ18Ewas cut with SphI in the multi-cloning site and was ligated with a DNAfragment obtained by cutting E. coli plasmid pBR322 (manufactured byTakara Shuzo Co. ) or pUC118 with SphI. The reaction mixture was used totransform E. coli TGl strain Δ(lac-pro)supE thi hsdD5/F' traD36proA+B+lacI^(q) lacZΔM15! (manufactured by Amersham Co.) by thewell-known calcium chloride method. The transformants were selected with500 μg Em/ml. They had a recombinant plasmid composed of pX3Δ18E andpBR322 or pUC118, as shown in FIG. 7 to FIG. 10 (pBR3Δ18E1, pBR3Δ18E2;p8X3×8E1, p8X3Δ18E2).

These transformants showed ampicillin resistance (50 μg/ml) derived fromE. coli pBR322 and pUC118 plasmids. These recombinant plasmids isolatedand purified from E. coli were used to transform Lc. lactis IL1403strain and transformants were selected with 25 μg/ml of Em as shown inExample 3. As a result, Em-resistant transformants were obtained. Theplasmids possessed by the transformants were prepared and therestriction enzyme-recognition sites were examined. As a result, theyeach had the same restriction endonuclease map as the map of thoseprepared from E. coli. From these results, pX3Δ18E, one of thederivatives of pBUL1, was found to be a useful shuttle vector if it isligated with an E. coli plasmid. For the transformants with such arecombinant plasmid could be selected with ampicillin and Em, or Em whenthe host was either E. coli or lactic acid, respectively. Thus, theusefulness of pBUL1 was further proved.

EXAMPLE 8

(Replication of pX3Δ18E Plasmid in E. coli):

The plasmid pX3Δ18E, obtained in Example 6, was prepared from thetransformant of Lc. lactis IL1403 by the method of reference 9, and wasused to transform E. coli TGl strain in the same manner as in Example 7.The thus obtained Em-resistant (Em 500 μg/ml) transformant had a plasmidhaving the same restriction endonuclease map as that of pX3Δ18E used forthe transformation. From the fact, it was found that the pX3Δ18Eplasmid, a derivative of pBUL1, is replicable even in E. coli, that E.coli with pX3Δ18E may be selected on the basis of the Em resistance andthat pX3Δ18E is useful as a shuttle vector between Gram-positivebacteria and Gram-negative bacteria. Thus, the usefulness of pBUL1 wasfurther proved.

References:

1. Holo, H. and I. F. Nes, (1989) Appl. Environ. Microbiol., 55 (12)3119-3123.

2. Mercenier, A., (1990) FEMS Microbiol. Rev., 87, 61-77.

3. Scheirlinck, T., et al., (1989) Appl. Environ. Microbiol., 55 (9)2130-2137.

4. Chassy, B. M. and J. L. Flickinger, (1987) FEMS Microbiol. Lett.,(44) 173-177.

5. de Vos, W. N., (1987) FEMS Microbiol. Rev., 46, 281-295.

6. Kok, J. et al., (1984) Appl. Environ. Microbiol., 48, 726-731.

7. Simon, D. and A. Chopin, (1988) Biochimie, 70 (4) 559-566.

8. Efthymiou, C., et at., (1962) J. Infect. Dis., 110, 258-267.

9. Anderson, D. and L. L. Mckay, (1983) Appl. Environ. Microbiol., 46,549-552.

10. Maniatis, T., et al., (1982) Molecular Cloning : A LaboratoryManual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.

11. LeBlanc, D. J. and L. N. Lee, (1984) J. Bacteriol., 157, 445-453.

12. Chang, S. and S. N. Cohen, (1979) Mol. GeL. Genet., 168, 111-115.

13. Yamane, K., et al., (1984) J. Biochem., 96, 1849-1858.

14. Yamane, K. (1987) "Kosoh-kin (Bacillus subtilis)", inBiseibutsu-gaku Kiso Kohza (Lectures on Fundamental Microbiology) (Ando,T. and Sakaguchi, K. (eds.)), pp. 168-215 (1987)

Advantage of the Invention

The species Lactobacillus delbrueckii is an industrially useful lacticacid bacterium, including subsp. bulgaricus and subsp. lactis which areimportant in production of and cheese and subsp. delbrueckii which isimportant in production of lactic acid.

However, with respect to Lactobacillus delbrueckii, neithertransformation nor the presence of an autonomously replicable plasmidhas heretofore been reported. The plasmid of the present invention isthe first reported up to now. Using the plasmid of the present inventionas a replicon, transformation of Lb. lactis, which is one of thisspecies, has become possible for the first time. Accordingly, it isexpected that various genes are inserted into the plasmid of the presentinvention to give recombinant plasmids which are introduced intosubspecies of Lactobacillus delbrueckii to construct improved strainshaving useful characteristics for example, a high lactose metablizingability or an improved milk protein degrading activity. In addition, thepresent invention provides a plasmid with a broad-host-range shuttlevector. Thus, the effectiveness of the present invention is furtherelevated.

Since the plasmid of the invention is one isolated from a strain of Lb.bulgaricus existing in yogurt, its safety has been confirmedhistorically. In addition, since the plasmid of the invention canreplicate not only in species of Lactobacillus delbrueckii but also inother bacteria important in the food industry, such as the generaBacillus and Lactococcus, it is expected to be useful not only in theproduction of various foods but also in breeding of micro-organismswhich produce various enzymes and physiologically active substances.

Reference to Deposited Microorganism under Rule 13-2:

1. Lactobacillus delbrueckii subsp. bulgaricus M-878

a. Name and Address of the Institution for Deposition of the presentMicroorganism:

Name: Fermentation Research Institute, Agency of Industrial Science andTechnology, Ministry of International Trade and Industry

Address: 1-3, Higashi 1-chome, Tsukuba-shi, ibaraki-ken, 305, Japan

b. Date of Deposition in the Institution stated in a: Jan. 29, 1991

c. Deposition Number rendered by the Institution stated in a: FERNBP-3758

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 1                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1344 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       CCCGGGGCGAAACGACATGGGGCGCTCAAACCATTGCTGAGGCGATCAATTACGTGCAAG60                CCCAGCATCCCGATCACGGCTATTTCCCAGCTCGCCAAAATTCCGGCATGAGGGTTGTTG120               AACCGGGTGAAAGAGCCACAGGCGAAACGCTTAGAATTACGATTGACGGACAGGAAAGAG180               AATTTCCGTTCAACGGCTTTTTCTATAACCGGGATTATGAAATGACCGAGGTTGGGTTTG240               CTAACAGGTTTGCCGATTGGTACGCCAAAGGGAAACTTGTTTATCACCCCGGCTTAAAGG300               CGTGGCTTATGTACAACCCAGAAACGGGGTCATGGATGCCGAATGAAGACGACAGACTGG360               GCAAGGATTTTAACCAGACCCCGGAAAAACTCATCGATAACTTGCGGATAAACCTCAAAT420               TTGAACAACCGCTATGGAAAAAAGTTGGGTTTAATCCCCAAAAGCCTAATAATCAAACAT480               TCGGGGAAAAGGCTTATGCTAGTGGCTATAGCCGGATCAGCACGGCCGCTGGGCAAAAGG540               CGACCCTTGAATTAGCTCAGAGCCGTTTAACCGTGCGTGCATTTAACGACTGTAAGACCG600               AGCTTAACACCCAGACAGGTTGGATTGACCTCAAAACGGGTGCTATTAGCCCTCACAACC660               CGGCGAAACTTTTTGACAAGGTCACGGATGCTGGTTTGCCTAATAAAGCCACAGAGGGTG720               ATGGCGGCAAGCTCTGGGATCGTTTCCTGAAAGAGACCTTTTGCGGCGATCTTGATTTGA780               TCGAGTTCGTACAGGCCTGCATAGGCTACAGCATTACGGGCAAAATCAATGAACAGGTCA840               TGTTTATCTGCAAAGGCAGTGGGGGCAACGGGAAGAGCATTTTTCTTGAATGCTTAAACG900               AGGTGCTGGGCGATTATAGCTCTGTTATCCCAATAGAAACGCTAACAGACAACGGCAAGG960               CTCAGCGTGACGGATCAGCACCAAGTCCAGACCTTGCAAGCCTTGAGGGCAAGCGGTTCG1020              TTATTACGAGCGAACCGAAAGAGCAGGTTACAATCGATGCTGGGACGGTCAAAACGGTGA1080              CGGGTGGCACTAAGTTAAAAGTTAGAATGCTACACCAAAACCCGATTGAGTTCCTGCCAC1140              AGTTTAAAATTTGGTGGCAATCTAACGGCTTGCCAAACGTCAACTTTAACGATTATGCTA1200              TTTTACGGCGCTTGATCGTCATCCCGTTTAAAAATGAGGTGCGAGAGGATGCGGTAGATA1260              TCAACCTCAAAAGCAAGCTAATGAAAGAGAAAGAGTTTATTTTAAAGTGGTGTGTTGAGG1320              GCGTGGCTAAATGGCAAGCCCGGG1344                                                  __________________________________________________________________________

We claim:
 1. A Lactobacillus delbrueckii transformant which has beentransformed with a recombinant plasmid comprising at least one foreigngene and a region necessary for replication of plasmid pBUL1, containingthe SmaI fragment of 1344 bp shown in SEQ ID NO:
 1. 2. The transformantaccording to claim 1 wherein the region necessary for replication is aDNA fragment shown by the thick line in FIG. 1, which is about 4 kbpbetween the position of the Eco47III site and the position about 1.1 kbpclockwise from the ScaI site and about 0.45 kbp counterclockwise fromthe NdeI site.
 3. The transformant according to claim 2 wherein saidbacterial transformants belong to Lactobacillus delbrueckii subsp.delbrueckii.
 4. The transformants according to claim 3 wherein theforeign gene is an L-lactate dehydrogenase gene.
 5. The transformantaccording to claim 2 wherein said bacterial transformants belong toLactobacillus delbrueckii subsp. bulgaricus.
 6. The transformantsaccording to claim 5 wherein the foreign gene is an L-lactatedehydrogenase gene.
 7. The transformant according to claim 2 whereinsaid bacterial transformants belong to Lactobacillus delbrueckii subsp.lactis.
 8. The transformant according to claim 7 wherein the foreigngene ms an L-lactate dehydrogenase gene.
 9. The transformant accordingto claim 2 wherein the foreign gene is an L-lactate dehydrogenase gene.10. The transformant according to claim 1 wherein said bacterialtransformants belong to Lactobacillus delbrueckii subsp. delbrueckii.11. The transformant according to claim 10 wherein the foreign gene isan L-lactate dehydrogenase gene.
 12. The transformant according to claim1 wherein said bacterial transformants belong to Lactobacillusdelbrueckii subsp. bulgaricus.
 13. The transformant according to claim12 wherein the foreign gene is an L-lactate dehydrogenase gene.
 14. Thetransformant according to claim 1 wherein said bacterial transformantsbelong to Lactobacillus delbrueckii subsp. lactis.
 15. The attransformant according to claim 14 wherein the foreign gene is anL-lactate dehydrogenase gene.
 16. The transformant according to claim 1wherein the foreign gene is an L-lactate dehydrogenase gens.